We have developed a fully automated bioreactor coupled to an on-line receptor affinity detection system. This analytical system provides detailed information on pharmacologically active metabolites of selective estrogen receptor modulators (SERMs) generated by cytochromes P450 (P450s). We demonstrated this novel concept by investigating the metabolic activation of tamoxifen and raloxifene by P450-containing pig and rat liver microsomes. The high resolution screening (HRS) system is based on the coupling of a P450-bioreactor to an HPLC-based estrogen receptor alpha (ERα) affinity assay. P450-derived metabolites of the SERMs were generated in the bioreactor, subsequently trapped on-line with solid phase extraction, and finally separated with gradient HPLC. Upon elution, the metabolites were screened on affinity for ERα with an on-line HRS assay. With this HRS system, we were able to follow, in a time-dependent manner, the formation of ERα-binding metabolites of tamoxifen and raloxifene. By analyzing the bioaffinity chromatograms with liquid chromatography-tandem mass spectrometry, structural information of the pharmacologically active metabolites was obtained as well. For tamoxifen, 15 active and 6 nonactive metabolites were observed, of which 5 were of primary, 10 of secondary, and 6 of an as yet unknown order of metabolism. Raloxifene was biotransformed in three primary and three secondary metabolites. MS/MS analysis revealed that three of the observed active metabolites of raloxifene were not described before. The present automated on-line HRS system coupled to a P450-containing bioreactor and an ERα-affinity detector proved very efficient, sensitive, and selective in metabolic profiling of SERMs. Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics.